Human Opsonins Induced during Meningococcal Disease Recognize Outer Membrane Proteins PorA and PorB

Abstract

Human opsonins directed against specific meningococcal outer membrane structures in sera obtained during meningococcal disease were quantified with a recently developed antigen-specific, opsonin-dependent phagocytosis and oxidative burst assay. Outer membrane vesicles (OMVs) and PorA (class 1) and PorB (class 3) proteins purified from mutants of the same strain (44/76; B:15:P1.7.16) were adsorbed to fluorescent beads, opsonized with acute- and convalescent-phase sera from 40 patients with meningococcal disease, and exposed to human leukocytes. Flow cytometric quantitation of the resulting leukocyte phagocytosis products (PPs) demonstrated that disease-induced serum opsonins recognized meningococcal OMV components and both porins. The PPPorA and PPPorB values induced by convalescent-phase sera correlated positively with the PPOMV values. However, the PPPorB values were higher than the PPPorA values in convalescent-phase sera (medians [ranges] of 754 [17 to 1,057] and 107 [4 to 458], respectively) (P < 0.0001) and correlated positively with higher levels of immunoglobulin G against PorB than against PorA as evaluated by enzyme-linked immunosorbent assay. Extensive individual variations in the anti-OMV and antiporin serum opsonic activities between patients infected by serotypes and serosubtypes homologous and heterologous to the target antigens were observed. Simultaneously measured oxidative burst activity correlated with the opsonophagocytosis, an indication that both of these important steps in the in vitro phagocytic elimination of meningococci are initiated by opsonins directed against OMV components, including PorA and PorB. In conclusion, human patient opsonins against meningococcal OMV components and in particular PorB epitopes were identified by this new method, which might facilitate selection of opsonin-inducing meningococcal antigens for inclusion in future vaccines.

Gingipain‐Specific IgG in the Sera of Patients With Periodontal Disease Is Necessary for Opsonophagocytosis of Porphyromonas gingivalis

“…In addition, a phagocytosis product (PP) was calculated for each sample by multiplying the % phagocytosis with the Pg/PMN. 39 Coupling Gingipains to Sepharose Beads and Adsorption of Human Sera Selective removal of P. gingivalis-and gingipainspecific antibodies from GAgP and control patient sera was accomplished by adsorption with either formaldehyde-fixed P. gingivalis or each gingipain linked to cyanogen-bromide activated sepharose-4B beads (SB). In brief, RgpA, RgpB, Kgp, and BSA (2 mg) were coupled separately to SB (0.2 mg) according to the manufacturer's instructions.…”

“…As phagocytosis of bacteria is not adequately described by either the percentage of PMNs actively taking up P. gingivalis or the number of P. gingivalis taken up by a participating PMN, we calculated a phagocytosis product to combine both of these attributes and obtain a better overall interpretation of phagocytosis. 39 P. gingivalis A7436 was opsonized with GAgP or control serum diluted 1:10 with human serum, as well as preimmune or immune rabbit sera as controls, human PMNs were added, aliquots of each reaction were taken at 15, 30, and 45 minutes, and the % phagocytosis, Pg/PMN, and PP were determined for each sample. Within 15 minutes there was an increase in the percentage of neutrophils taking up P. gingivalis opsonized with GAgP sera compared to control sera (Table 1) and a concomitant increase in the number of Pg/PMN opsonized with GAgP patient serum.…”

Neisseria meningitidis serogroup B: laboratory correlates of protection

“…FITC-labeling or heat-killing of bacteria might destroy epitope conformation and therefore not re£ect the epitope exposition on live bacteria [31]. Antigen-coated beads probably present antigens similar to the native conformation and have the advantage that antigen speci¢city of serum opsonic activity can be studied as well [22,32]. Serum opsonic activity has been shown to correlate with IgG titers against OMVs, PorA and PorB measured by enzyme-linked immunosorbent assay (ELISA) [22,33] and SBA [34].…”

“…Antigen-coated beads probably present antigens similar to the native conformation and have the advantage that antigen speci¢city of serum opsonic activity can be studied as well [22,32]. Serum opsonic activity has been shown to correlate with IgG titers against OMVs, PorA and PorB measured by enzyme-linked immunosorbent assay (ELISA) [22,33] and SBA [34]. The measurement of serum opsonic activity is expected to be of additional value to survey a vaccine trial, although so far it has not been tested in any e⁄cacy trial yet.…”

Human Opsonins Induced during Meningococcal Disease Recognize Transferrin Binding Protein Complexes

“…Whereas the majority of studies concerning the immune response following meningococcal disease and vaccination have focused on the role of human serum bactericidal activity against meningococci, some reports indicate that phagocytic elimination of meningococci is an important host defense mechanism (7,26,28,30) which is stimulated by serum opsonins (complement and antibodies). Increasing serum opsonic activity against meningococci has been demonstrated during meningococcal disease (18,19), and disease-induced opsonins have been shown to recognize meningococcal outer membrane components PorA and PorB (23).…”